Effective preselection of sex has been done in various species of livestock and domestic animals using the flow cytometry. A guaranteed high sorting accuracy is a key performance for the widespread use of sperm sexing. However, the sexing technique needed to be validated to ensure the accuracy of the technology.
To be able to specifically produce either male or female offspring in the dog, we developed a technique to determine the sex of canine spermatozoa using SYBR® Green real-time quantitative PCR (qPCR).
Two sets of primers, ZFX and SRY, were designed specifically for X- and Y- chromosome canine genes, respectively. Plasmid was inserted with ZFX and SRY gene fragment separately to create standard curves that ranged from 300 to 3,000,000 copies. In canine genome, both ZFX and SRY genes exist as single copy number and are commonly used as a DNA marker in sex determination.
Real-time qPCR of bovine spermatozoa DNA samples and cloned plasmid ZFX and SRY genes for creating standard samples were performed simultaneously. There was no significant difference in percentages between the theoretical ratio (1:1) and unsexed X- and Y- chromosome-bearing canine spermatozoa in any of the nine dogs. In addition, the mean purities of sorted sex chromosomes in spermatozoa of the nine dogs were 91% for the X chromosome fraction and 90% for the Y chromosome fraction.
This technique was a rapid and reliable technique in quantifying the sex ratio of X- and Y- chromosome-bearing spermatozoa in semen sample.